Abstract

The Hedgehog proteins are potent organizers of animal development. They carry a cholesterol ester at the C terminus of their signaling domain. The membrane anchoring mediated by this lipophilic modification was studied by means of an approach integrating cell biology, biochemistry, biophysics, and organic chemistry techniques. Sterol-modified and fluorescent-labeled Hedgehog-derived peptides and proteins were synthesized and investigated in biophysical and cell-biological assays. These experiments revealed that cholesterol alone anchors proteins to membranes with significant strength and half-times for spontaneous desorption of several hours. Its membrane anchoring ability is comparable to dual lipidation motifs such as double geranylgeranylation or S-palmitoylation plus S-farnesylation found in other lipidated proteins. The experiments also demonstrate that membrane binding changes dramatically if short lipidated peptides are equipped with a large protein. These data suggest that for Hedgehog release and subsequent signaling an interaction partner such as the Dispatched protein is necessary. In addition to these findings the described approach allows one to correlate biophysical data obtained with model peptides with data determined with fully functional proteins and to combine results from in vitro and in vivo experiments. It should be generally applicable to other membrane anchors and proteins.

Membrane binding of lipopeptides. (A) Intervesicle transfer experiments using sterol-modified peptides 7 (gray trace) and 8 (black trace). POPC vesicles containing 2 mol % Rho-DHPE and either 1 mol % peptide 7 or 8 were diluted in buffer to a final concentration of 50 nM NBD-lipopeptide and mixed with a 12-fold excess of pure POPC vesicles (arrow indicating time of addition). The change in fluorescence was monitored at 535 nm. All experiments were carried out at 20°C. (B) Examination of the flip-flop-exchange of the lipopeptides 7 (gray) and 8 (black). For this assay POPC vesicles loaded only with 1 mol % NBD-labeled lipopetide 7 or 8 were diluted to a final concentration of 50 nM lipopeptide. After a stable baseline had been established, 10 mM sodium dithionite was added (arrow). All experiments were carried out at 20°C. The change in fluorescence was monitored at 535 nm.

Titration of lipoproteins 11 and 12 with POPC/Rho-DHPE vesicles. Androstenol- and cholesterol-modified lipoproteins were diluted in buffer to a final concentration between 50 and 100 nM and the change in NBD emission at 535 nm was monitored after stepwise addition of POPC vesicles containing 4 mol % Rho-DHPE. (A) Raw data for titration of lipoprotein 11 (♦, left axis). Nonspecific increase in signal due to the addition of Rho-DHPE-containing vesicles was measured in a cuvette filled with buffer only (+, right axis). (B and C) Binding curves for lipoprotein 11 and 12, respectively.